To explore the distinct genotypic and phenotypic states of melanoma tumors we applied single-cell RNA-seq to 4,645 single cells isolated from 19 patients, profiling malignant, immune, stromal and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that “MITF-high” tumors also contained “AXL-high” tumor cells. Single-cell analyses suggested distinct tumor micro-environmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and to clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single cell genomics offers insights with implications for both targeted and immune therapies.

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SUMMARY Exhausted CD8+ T cells function poorly and are negatively regulated by inhibitory receptors. Transcriptional profiling has identified gene expression changes associated with exhaustion. However, the transcriptional pathways critical to the differences between exhausted and functional memory CD8+ T cells are unclear. We thus defined transcriptional coexpression networks to define pathways centrally involved in exhaustion versus memory. These studies revealed differences between exhausted and memory CD8+ T cells including the following: lack of coordinated transcriptional modules of quiescence during exhaustion, centrally connected hub genes, pathways such as transcription factors, genes involved in regulation of immune responses, and DNA repair genes, as well as differential connectivity for genes including T-bet, Eomes, and other transcription factors. These data identify pathways involved in CD8+ T cell exhaustion, and highlight the context-dependent nature of transcription factors in exhaustion versus memory.

The development and severity of inflammatory bowel diseases (IBD) and other chronic inflammatory conditions can be influenced by host genetic and environmental factors, including signals derived from commensal bacteria1–6. However, the mechanisms that integrate these diverse cues remain undefined. Here we demonstrate that mice with an intestinal epithelial cell-specific deletion of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3ΔIEC mice) exhibited extensive dysregulation of IEC-intrinsic gene expression, including decreased basal expression of genes associated with antimicrobial defense. Critically, conventionally-housed HDAC3ΔIEC mice demonstrated loss of Paneth cells, impaired IEC function and alterations in the composition of intestinal commensal bacteria. In addition, HDAC3ΔIEC mice exhibited significantly increased susceptibility to intestinal damage and inflammation, indicating that epithelial expression of HDAC3 plays a central role in maintaining intestinal homeostasis. Rederivation of HDAC3ΔIEC mice into germ-free conditions revealed that dysregulated IEC gene expression, Paneth cell homeostasis, and intestinal barrier function were largely restored in the absence of commensal bacteria. While the specific mechanisms through which IEC-intrinsic HDAC3 expression regulates these complex phenotypes remain to be elucidated, these data indicate that HDAC3 is a critical factor that integrates commensal bacteria-derived signals to calibrate epithelial cell responses required to establish normal host-commensal relationships and maintain intestinal homeostasis.

Interleukin-25 preferentially elicits multipotent progenitor type 2 cells, which are distinct from other populations of type 2 innate lymphoid cells.

We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1™ system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1045-6) contains supplementary material, which is available to authorized users.

Evaluation of myeloid-derived suppressor cells (MDSC), a cell type implicated in T-cell suppression, may inform immune status. However, a uniform methodology is necessary for prospective testing as a biomarker. We report the use of a computational algorithm-driven analysis of whole blood and cryopreserved samples for monocytic MDSC (m-MDSC) quantity that removes variables related to blood processing and user definitions. Applying these methods to samples from melanoma patients identifies differing frequency distribution of m-MDSC relative to that in healthy donors (HD). Patients with a pre-treatment m-MDSC frequency outside a preliminary definition of HD range (<14.9%) were significantly more likely to achieve prolonged overall survival following treatment with ipilimumab, an antibody that promotes T-cell activation and proliferation. m-MDSC frequencies inversely correlated with peripheral CD8+ T-cell expansion following ipilimumab. Algorithm-driven analysis may enable not only development of a novel pre-treatment biomarker for ipilimumab therapy, but also prospective validation of peripheral blood m-MDSC as a biomarker in multiple disease settings.

SUMMARY Extramedullary hematopoiesis (EMH) refers to the differentiation of hematopoietic stem cells (HSCs) into effector cells that occurs in compartments outside of the bone marrow. Previous studies linked pattern recognition receptor (PRR)-expressing HSCs, EMH and immune responses to microbial stimuli. However, whether EMH operates in broader immune contexts remains unknown. Here, we demonstrate a previously unrecognized role for thymic stromal lymphopoietin (TSLP) in promoting the population expansion of progenitor cells in the periphery and identify that TSLP-elicited progenitors differentiated into effector cells including macrophages, dendritic cells and granulocytes; and that these cells contributed to type 2 cytokine responses. The frequency of circulating progenitor cells was also increased in allergic patients with a gain-of-function polymorphism in TSLP, suggesting the TSLP-EMH pathway may operate in human disease. These data identify that TSLP-induced EMH contributes to the development of allergic inflammation and indicate that EMH is a conserved mechanism of innate immunity.